1. Field of the Invention
The invention relates to fluorescent labels useful for chemical and biotechnological applications.
2. Background of the Invention
Fluorescent labels are widely applied in biology, biotechnology, and medicine. In addition, fluorophores are becoming increasingly useful in combinatorial chemistry and biology, both as encoders of library members and as reporters of chemical reactions.
Nucleic acids can be fluorescently tagged either enzymatically or synthetically. Enzymatic incorporation is carried out by use of nucleoside triphosphates carrying a given fluorophore. Incorporation into DNA by chemical methods is especially common. Chemical methods of incorporating fluorescent reagents into DNA is done by either of two methods: direct incorporation of a label which has been converted to a phosphoramidite derivative or incorporation of an amine into the oligonucleotide, followed by later derivatization with a fluorophore isothiocyanate or NHS ester derivative.
One way to generate new fluorescence properties is to encourage two dyes to interact photophysically. A prominent and useful example of this is FRET dye pairs, which have been instrumental in decoding the human genome (Glazer, et al. Curr. Opin. Biotechnol. 8: 94-102, 1997). Electronically interacting fluorophores can display new properties that individual dyes do not have, including increased Stokes shifts, specifically tuned excitations, or increased brightness.
Up to three commercial dyes have been used in DNA-based FRET libraries, generating up to 16 different molecules spaced by 1-2 dozen natural nucleosides (Kawahara, et al., Chem. Commun. 563-564, 1999; Tong, et al., Nature Biotech. 19: 756-759, 2001; Tong, et al., J. Am. Chem. Soc. 123: 12923-12924, 2001; Tong, et al., Nucleic Acids Res. 30: e19, 2002). These libraries used only commercially available fluorophores, and used only the FRET mechanism of energy transfer.
U.S. Pat. No. 6,218,108 B1 (issued Apr. 17, 2001) and U.S. Pat. No. 6,140,480 (issued Oct. 31, 2000) disclose nucleoside analogs with polycyclic aromatic groups attached. The aromatic groups include anthracene, phenanthrene, pyrene, stilbene, tetracene, pentacene, and derivatives thereof. The aromatic hydrocarbon groups are attached to the C-1 (1′) carbon of the sugar moiety in a nucleotide or nucleoside. The fluorescent nucleosides can be incorporated into nucleic acids. The nucleic acids are suggested as being useful as probes for targets in tissues, in solution, or immobilized on membranes. Sequences are disclosed for oligonucleotides containing up to seven adjacent pyrene molecules.
The preparation of and incorporation of porphyrin C-nucleosides into DNA was disclosed on the internet on Nov. 19, 2002 (Organic Lett. 4(25): 4377-4380 (2002)). A porphyrin was coupled with 2-deoxy-D-ribose and incorporated into DNA using phosphoramidite chemistry. Substitution at the ends of a 5′-modified self-complementary duplex was found to be thermally and thermodynamically stabilizing. The porphyrin strongly intercalated into the duplex when located near the center, and retained its fluorescence properties in DNA.
As a result of the greatly expanding use of fluorescent labels in research and diagnostic applications, there is a corresponding increase in the need for new fluorophores having a wider range of spectral characteristics.